RESUMO
The HIV-1 capsid is composed of capsid (CA) protein hexamers and pentamers (capsomers) that contain a central pore hypothesised to regulate capsid assembly and facilitate nucleotide import early during post-infection. These pore functions are mediated by two positively charged rings created by CA Arg-18 (R18) and Lys-25 (K25). Here we describe the forced evolution of viruses containing mutations in R18 and K25. Whilst R18 mutants fail to replicate, K25A viruses acquire compensating mutations that restore nearly wild-type replication fitness. These compensating mutations, which rescue reverse transcription and infection without reintroducing lost pore charges, map to three adaptation hot-spots located within and between capsomers. The second-site suppressor mutations act by restoring the formation of pentamers lost upon K25 mutation, enabling closed conical capsid assembly both in vitro and inside virions. These results indicate that there is no intrinsic requirement for K25 in either nucleotide import or capsid assembly. We propose that whilst HIV-1 must maintain a precise hexamer:pentamer equilibrium for proper capsid assembly, compensatory mutations can tune this equilibrium to restore fitness lost by mutation of the central pore.
Assuntos
Proteínas do Capsídeo , Capsídeo , HIV-1 , Mutação , Montagem de Vírus , Replicação Viral , HIV-1/genética , HIV-1/fisiologia , Montagem de Vírus/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/química , Capsídeo/metabolismo , Humanos , Replicação Viral/genética , Vírion/metabolismo , Vírion/genética , Células HEK293 , Infecções por HIV/virologia , Infecções por HIV/genéticaRESUMO
We explored how a simple retrovirus, Mason-Pfizer monkey virus (M-PMV) to facilitate its replication process, utilizes DHX15, a cellular RNA helicase, typically engaged in RNA processing. Through advanced genetic engineering techniques, we showed that M-PMV recruits DHX15 by mimicking cellular mechanisms, relocating it from the nucleus to the cytoplasm to aid in viral assembly. This interaction is essential for the correct packaging of the viral genome and critical for its infectivity. Our findings offer unique insights into the mechanisms of viral manipulation of host cellular processes, highlighting a sophisticated strategy that viruses employ to leverage cellular machinery for their replication. This study adds valuable knowledge to the understanding of viral-host interactions but also suggests a common evolutionary history between cellular processes and viral mechanisms. This finding opens a unique perspective on the export mechanism of intron-retaining mRNAs in the packaging of viral genetic information and potentially develop ways to stop it.
Assuntos
Vírus dos Macacos de Mason-Pfizer , RNA Viral , Montagem de Vírus , Animais , Humanos , Núcleo Celular/metabolismo , Núcleo Celular/virologia , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Genoma Viral , Células HEK293 , Vírus dos Macacos de Mason-Pfizer/genética , Vírus dos Macacos de Mason-Pfizer/metabolismo , Vírus dos Macacos de Mason-Pfizer/fisiologia , RNA Helicases/metabolismo , RNA Helicases/genética , RNA Viral/metabolismo , RNA Viral/genética , Montagem de Vírus/genética , Montagem de Vírus/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
HIV-1 transcript function is controlled in part by twinned transcriptional start site usage, where 5' capped RNAs beginning with a single guanosine (1G) are preferentially packaged into progeny virions as genomic RNA (gRNA) whereas those beginning with three sequential guanosines (3G) are retained in cells as mRNAs. In 3G transcripts, one of the additional guanosines base pairs with a cytosine located within a conserved 5' polyA element, resulting in formation of an extended 5' polyA structure as opposed to the hairpin structure formed in 1G RNAs. To understand how this remodeling influences overall transcript function, we applied in vitro biophysical studies with in-cell genome packaging and competitive translation assays to native and 5' polyA mutant transcripts generated with promoters that differentially produce 1G or 3G RNAs. We identified mutations that stabilize the 5' polyA hairpin structure in 3G RNAs, which promote RNA dimerization and Gag binding without sequestering the 5' cap. None of these 3G transcripts were competitively packaged, confirming that cap exposure is a dominant negative determinant of viral genome packaging. For all RNAs examined, conformations that favored 5' cap exposure were both poorly packaged and more efficiently translated than those that favored 5' cap sequestration. We propose that structural plasticity of 5' polyA and other conserved RNA elements place the 5' leader on a thermodynamic tipping point for low-energetic (~3 kcal/mol) control of global transcript structure and function.
Assuntos
Genoma Viral , HIV-1 , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Viral , HIV-1/genética , RNA Viral/genética , RNA Viral/metabolismo , RNA Viral/química , Humanos , Empacotamento do Genoma Viral , Mutação , Montagem de Vírus/genética , Capuzes de RNA/metabolismo , Capuzes de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Novel bat H17N10 and H18N11 influenza A viruses (IAVs) are incapable of reassortment with conventional IAVs during co-infection. To date, the underlying mechanisms that inhibit bat and conventional IAV reassortment remain poorly understood. Herein, we used the bat influenza M gene in the PR8 H1N1 virus genetic background to determine the molecular basis that restricts reassortment of segment 7. Our results showed that NEP and M1 from bat H17N10 and H18N11 can interact with PR8 M1 and NEP, resulting in mediating PR8 viral ribonucleoprotein (vRNP) nuclear export and formation of virus-like particles with single vRNP. Further studies demonstrated that the incompatible packaging signals (PSs) of H17N10 or H18N11 M segment led to the failure to rescue recombinant viruses in the PR8 genetic background. Recombinant PR8 viruses (rPR8psH18M and rPR8psH17M) containing bat influenza M coding region flanked with the PR8 M PSs were rescued but displayed lower replication in contrast to the parental PR8 virus, which is due to a low efficiency of recombinant virus uncoating correlating with the functions of the bat M2. Our studies reveal molecular mechanisms of the M gene that hinder reassortment between bat and conventional IAVs, which will help to understand the biology of novel bat IAVs. IMPORTANCE: Reassortment is one of the mechanisms in fast evolution of influenza A viruses (IAVs) and responsible for generating pandemic strains. To date, why novel bat IAVs are incapable of reassorting with conventional IAVs remains completely understood. Here, we attempted to rescue recombinant PR8 viruses with M segment from bat IAVs to understand the molecular mechanisms in hindering their reassortment. Results showed that bat influenza NEP and M1 have similar functions as respective counterparts of PR8 to medicating viral ribonucleoprotein nuclear export. Moreover, the incompatible packaging signals of M genes from bat and conventional IAVs and impaired bat M2 functions are the major reasons to hinder their reassortment. Recombinant PR8 viruses with bat influenza M open reading frames were generated but showed attenuation, which correlated with the functions of the bat M2 protein. Our studies provide novel insights into the molecular mechanisms that restrict reassortment between bat and conventional IAVs.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Vírus Reordenados , Humanos , Vírus Reordenados/genética , Animais , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Quirópteros/virologia , Proteínas da Matriz Viral/metabolismo , Proteínas da Matriz Viral/genética , Influenza Humana/virologia , Influenza Humana/metabolismo , Células HEK293 , Replicação Viral , Montagem de Vírus/genética , Células Madin Darby de Rim Canino , Cães , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genéticaRESUMO
DNA recognition is critical for assembly of double-stranded DNA viruses, particularly for the initiation of packaging the viral genome into the capsid. The key component that recognizes viral DNA is the small terminase protein. Despite prior studies, the molecular mechanism for DNA recognition remained elusive. Here, we address this question by identifying the minimal site in the bacteriophage HK97 genome specifically recognized by the small terminase and determining the structure of this complex by cryoEM. The circular small terminase employs an entirely unexpected mechanism in which DNA transits through the central tunnel, and sequence-specific recognition takes place as it emerges. This recognition stems from a substructure formed by the N- and C-terminal segments of two adjacent protomers which are unstructured when DNA is absent. Such interaction ensures continuous engagement of the small terminase with DNA, enabling it to slide along the DNA while simultaneously monitoring its sequence. This mechanism allows locating and instigating packaging initiation and termination precisely at the specific cos sequence.
Assuntos
DNA Viral , Genoma Viral , DNA Viral/genética , DNA Viral/metabolismo , DNA Viral/química , Microscopia Crioeletrônica , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Modelos Moleculares , Empacotamento do DNA , Montagem de Vírus/genética , Bacteriófagos/genética , Empacotamento do Genoma ViralRESUMO
A critical part of the hepatitis B virus (HBV) life cycle is the packaging of the pregenomic RNA (pgRNA) into nucleocapsids. While this process is known to involve several viral elements, much less is known about the identities and roles of host proteins in this process. To better understand the role of host proteins, we isolated pgRNA and characterized its protein interactome in cells expressing either packaging-competent or packaging-incompetent HBV genomes. We identified over 250 host proteins preferentially associated with pgRNA from the packaging-competent version of the virus. These included proteins already known to support capsid formation, enhance viral gene expression, catalyze nucleocapsid dephosphorylation, and bind to the viral genome, demonstrating the ability of the approach to effectively reveal functionally significant host-virus interactors. Three of these host proteins, AURKA, YTHDF2, and ATR, were selected for follow-up analysis. RNA immunoprecipitation qPCR (RIP-qPCR) confirmed pgRNA-protein association in cells, and siRNA knockdown of the proteins showed decreased encapsidation efficiency. This study provides a template for the use of comparative RNA-protein interactome analysis in conjunction with virus engineering to reveal functionally significant host-virus interactions.
Assuntos
Vírus da Hepatite B , RNA Viral , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Humanos , RNA Viral/genética , RNA Viral/metabolismo , Genoma Viral , Interações Hospedeiro-Patógeno/genética , Empacotamento do Genoma Viral/genética , Montagem de Vírus/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNARESUMO
The 3' untranslated region (3'UTR) of the hepatitis C virus (HCV) RNA genome, which contains a highly conserved 3' region named the 3'X-tail, plays an essential role in RNA replication and promotes viral IRES-dependent translation. Although our previous work has found a cis-acting element for genome encapsidation within 3'X, there is limited information on the involvement of the 3'UTR in particle formation. In this study, proteomic analyses identified host cell proteins that bind to the 3'UTR containing the 3'X region but not to the sequence lacking the 3'X. Further characterization showed that RNA-binding proteins, ribosomal protein L17 (RPL17), and Y-box binding protein 1 (YBX1) facilitate the efficient production of infectious HCV particles in the virus infection cells. Using small interfering RNA (siRNA)-mediated gene silencing in four assays that distinguish between the various stages of the HCV life cycle, RPL17 and YBX1 were found to be most important for particle assembly in the trans-packaging assay with replication-defective subgenomic RNA. In vitro assays showed that RPL17 and YBX1 bind to the 3'UTR RNA and deletion of the 3'X region attenuates their interaction. Knockdown of RPL17 or YBX1 resulted in reducing the amount of HCV RNA co-precipitating with the viral Core protein by RNA immunoprecipitation and increasing the relative distance in space between Core and double-stranded RNA by confocal imaging, suggesting that RPL17 and YBX1 potentially affect HCV RNA-Core interaction, leading to efficient nucleocapsid assembly. These host factors provide new clues to understanding the molecular mechanisms that regulate HCV particle formation. IMPORTANCE: Although basic research on the HCV life cycle has progressed significantly over the past two decades, our understanding of the molecular mechanisms that regulate the process of particle formation, in particular encapsidation of the genome or nucleocapsid assembly, has been limited. We present here, for the first time, that two RNA-binding proteins, RPL17 and YBX1, bind to the 3'X in the 3'UTR of the HCV genome, which potentially acts as a packaging signal, and facilitates the viral particle assembly. Our study revealed that RPL17 and YBX1 exert a positive effect on the interaction between HCV RNA and Core protein, suggesting that the presence of both host factors modulate an RNA structure or conformation suitable for packaging the viral genome. These findings help us to elucidate not only the regulatory mechanism of the particle assembly of HCV but also the function of host RNA-binding proteins during viral infection.
Assuntos
Regiões 3' não Traduzidas , Genoma Viral , Hepacivirus , RNA Viral , Proteínas Ribossômicas , Montagem de Vírus , Proteína 1 de Ligação a Y-Box , Regiões 3' não Traduzidas/genética , Hepacivirus/genética , Hepacivirus/fisiologia , Hepacivirus/metabolismo , Humanos , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Proteína 1 de Ligação a Y-Box/metabolismo , Proteína 1 de Ligação a Y-Box/genética , Montagem de Vírus/genética , RNA Viral/metabolismo , RNA Viral/genética , Replicação Viral , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteômica/métodosRESUMO
Genetic screens have transformed our ability to interrogate cellular factor requirements for viral infections1,2, but most current approaches are limited in their sensitivity, biased towards early stages of infection and provide only simplistic phenotypic information that is often based on survival of infected cells2-4. Here, by engineering human cytomegalovirus to express single guide RNA libraries directly from the viral genome, we developed virus-encoded CRISPR-based direct readout screening (VECOS), a sensitive, versatile, viral-centric approach that enables profiling of different stages of viral infection in a pooled format. Using this approach, we identified hundreds of host dependency and restriction factors and quantified their direct effects on viral genome replication, viral particle secretion and infectiousness of secreted particles, providing a multi-dimensional perspective on virus-host interactions. These high-resolution measurements reveal that perturbations altering late stages in the life cycle of human cytomegalovirus (HCMV) mostly regulate viral particle quality rather than quantity, establishing correct virion assembly as a critical stage that is heavily reliant on virus-host interactions. Overall, VECOS facilitates systematic high-resolution dissection of the role of human proteins during the infection cycle, providing a roadmap for in-depth study of host-herpesvirus interactions.
Assuntos
Sistemas CRISPR-Cas , Infecções por Citomegalovirus , Citomegalovirus , Interações Hospedeiro-Patógeno , RNA Guia de Sistemas CRISPR-Cas , Replicação Viral , Humanos , Linhagem Celular , Sistemas CRISPR-Cas/genética , Citomegalovirus/genética , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/virologia , Genoma Viral/genética , Interações Hospedeiro-Patógeno/genética , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Vírion/genética , Vírion/metabolismo , Montagem de Vírus/genética , Liberação de Vírus/genética , Replicação Viral/genéticaRESUMO
HIV-1 Gag polyprotein plays a pivotal role in assembly and budding of new particles, by specifically packaging two copies of viral gRNA in the host cell cytoplasm and selecting the cell plasma membrane for budding. Both gRNA and membrane selections are thought to be mediated by the compact form of Gag. This compact form binds to gRNA through both its matrix (MA) and nucleocapsid (NC) domains in the cytoplasm. At the plasma membrane, the membrane competes with gRNA for Gag binding, resulting in a transition to the extended form of Gag found in immature particles with MA bound to membrane lipids and NC to gRNA. The Gag compact form was previously evidenced in vitro. Here, we demonstrated the compact form of Gag in cells by confocal microscopy, using a bimolecular fluorescence complementation approach with a split-GFP bipartite system. Using wild-type Gag and Gag mutants, we showed that the compact form is highly dependent on the binding of MA and NC domains to RNA, as well as on interactions between MA and CA domains. In contrast, Gag multimerization appears to be less critical for the accumulation of the compact form. Finally, mutations altering the formation of Gag compact form led to a strong reduction in viral particle production and infectivity, revealing its key role in the production of infectious viral particles.
Assuntos
HIV-1 , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana , HIV-1/metabolismo , HIV-1/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Montagem de Vírus/genética , Humanos , Vírion/metabolismo , Vírion/genética , Ligação Proteica , RNA Viral/metabolismo , RNA Viral/genética , Membrana Celular/metabolismo , Membrana Celular/virologiaRESUMO
How do segmented RNA viruses correctly recruit their genome has yet to be clarified. Bluetongue virus is a double-stranded RNA virus with 10 segments of different sizes, but it assembles its genome in single-stranded form through a series of specific RNA-RNA interactions prior to packaging. In this study, we determined the structure of each BTV transcript, individually and in different combinations, using 2'-hydroxyl acylation analysed by primer extension and mutational profiling (SHAPE-MaP). SHAPE-MaP identified RNA structural changes during complex formation and putative RNA-RNA interaction sites. Our data also revealed a core RNA-complex of smaller segments which serves as the foundation ('anchor') for the assembly of a complete network composed of ten ssRNA segments. The same order of core RNA complex formation was identified in cells transfected with viral RNAs. No viral protein was required for these assembly reactions. Further, substitution mutations in the interacting bases within the core assemblies, altered subsequent segment addition and affected virus replication. These data identify a wholly RNA driven reaction that may offer novel opportunities for designed attenuation or antiviral therapeutics.
Assuntos
Vírus Bluetongue , Genoma Viral , RNA Viral , Replicação Viral , Vírus Bluetongue/genética , RNA Viral/metabolismo , RNA Viral/genética , RNA Viral/química , Animais , Replicação Viral/genética , Conformação de Ácido Nucleico , Montagem de Vírus/genética , Linhagem Celular , MutaçãoRESUMO
Positive-strand RNA [(+)RNA] viruses include pandemic SARS-CoV-2, tumor-inducing hepatitis C virus, debilitating chikungunya virus (CHIKV), lethal encephalitis viruses, and many other major pathogens. (+)RNA viruses replicate their RNA genomes in virus-induced replication organelles (ROs) that also evolve new viral species and variants by recombination and mutation and are crucial virus control targets. Recent cryo-electron microscopy (cryo-EM) reveals that viral RNA replication proteins form striking ringed 'crowns' at RO vesicle junctions with the cytosol. These crowns direct RO vesicle formation, viral (-)RNA and (+)RNA synthesis and capping, innate immune escape, and transfer of progeny (+)RNA genomes into translation and encapsidation. Ongoing studies are illuminating crown assembly, sequential functions, host factor interactions, etc., with significant implications for control and beneficial uses of viruses.
Assuntos
Genoma Viral , Organelas , RNA Viral , Replicação Viral , Replicação Viral/genética , Humanos , Genoma Viral/genética , Organelas/virologia , Organelas/genética , Organelas/ultraestrutura , RNA Viral/genética , Vírus de RNA de Cadeia Positiva/genética , Microscopia Crioeletrônica , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Montagem de Vírus/genética , Compartimentos de Replicação Viral , AnimaisRESUMO
Poxvirus assembly has been an intriguing area of research for several decades. While advancements in experimental techniques continue to yield fresh insights, many questions are still unresolved. Large genome sizes of up to 380 kbp, asymmetrical structure, an exterior lipid bilayer, and a cytoplasmic life cycle are some notable characteristics of these viruses. Inside the particle are two lateral bodies and a protein wall-bound-biconcave core containing the viral nucleocapsid. The assembly progresses through five major stages-endoplasmic reticulum (ER) membrane alteration and rupture, crescent formation, immature virion formation, genome encapsidation, virion maturation and in a subset of viruses, additional envelopment of the virion prior to its dissemination. Several large dsDNA viruses have been shown to follow a comparable sequence of events. In this chapter, we recapitulate our understanding of the poxvirus morphogenesis process while reviewing the most recent advances in the field. We also briefly discuss how virion assembly aids in our knowledge of the evolutionary links between poxviruses and other Nucleocytoplasmic Large DNA Viruses (NCLDVs).
Assuntos
Poxviridae , Montagem de Vírus , Poxviridae/genética , Poxviridae/fisiologia , Montagem de Vírus/genética , Humanos , Genoma Viral , Vírion/genética , Vírion/ultraestrutura , Animais , Evolução Molecular , Retículo Endoplasmático/virologiaRESUMO
Pseudorabies virus (PRV) is recognized as the aetiological agent responsible for Aujeszky's disease, or pseudorabies, in swine populations. Rab6, a member of the small GTPase family, is implicated in various membrane trafficking processes, particularly exocytosis regulation. Its involvement in PRV infection, however, has not been documented previously. In our study, we observed a significant increase in the Rab6 mRNA and protein levels in both PK-15 porcine kidney epithelial cells and porcine alveolar macrophages, as well as in the lungs and spleens of mice infected with PRV. The overexpression of wild-type Rab6 and its GTP-bound mutant facilitated PRV proliferation, whereas the GDP-bound mutant form of Rab6 had no effect on viral propagation. These findings indicated that the GTPase activity of Rab6 was crucial for the successful spread of PRV. Further investigations revealed that the reduction in Rab6 levels through knockdown significantly hampered PRV proliferation and disrupted virus assembly and egress. At the molecular level, Rab6 was found to interact with the PRV glycoproteins gB and gE, both of which are essential for viral assembly and egress. Our results collectively suggest that PRV exploits Rab6 to expedite its assembly and egress and identify Rab6 as a promising novel target for therapeutic treatment for PRV infection.
Assuntos
Herpesvirus Suídeo 1 , Montagem de Vírus , Liberação de Vírus , Proteínas rab de Ligação ao GTP , Animais , Camundongos , Linhagem Celular , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/metabolismo , Pseudorraiva/virologia , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Suínos , Doenças dos Suínos/virologia , Montagem de Vírus/genética , Liberação de Vírus/genéticaRESUMO
The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38 ± 10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to ancestral and mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining nucleocapsid protein variants in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multivalent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.
Assuntos
Proteínas do Nucleocapsídeo de Coronavírus , Multimerização Proteica , RNA Viral , SARS-CoV-2 , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/química , Proteínas do Nucleocapsídeo de Coronavírus/química , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/genética , RNA Viral/metabolismo , RNA Viral/química , RNA Viral/genética , Ligação Proteica , Sítios de Ligação , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Montagem de Vírus/genética , Humanos , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , Proteínas do Nucleocapsídeo/genética , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , COVID-19/virologiaRESUMO
Influenza A virus contains regions of its segmented genome associated with ability to package the segments into virions, but many such regions are poorly characterised. We provide detailed predictions of the key locations within these packaging-associated regions, and their structures, by applying a recently-improved pipeline for delineating constrained regions in RNA viruses and applying structural prediction algorithms. We find and characterise other known constrained regions within influenza A genomes, including the region associated with the PA-X frameshift, regions associated with alternative splicing, and constraint around the initiation motif for a truncated PB1 protein, PB1-N92, associated with avian viruses. We further predict the presence of constrained regions that have not previously been described. The extra characterisation our work provides allows investigation of these key regions for drug target potential, and points towards determinants of packaging compatibility between segments.
Assuntos
Biologia Computacional , Vírus da Influenza A , Montagem de Vírus , Vírus da Influenza A/genética , Montagem de Vírus/genética , Biologia Computacional/métodos , Genoma Viral/genética , Algoritmos , Simulação por Computador , RNA Viral/genética , RNA Viral/química , RNA Viral/metabolismo , Humanos , Vírus de RNA/genéticaRESUMO
BACKGROUND: It is generally believed that hepatitis B virus (HBV) core protein (HBc) dephosphorylation (de-P) is important for viral DNA synthesis and virion secretion. HBV polymerase contains four domains for terminal protein, spacer, reverse transcriptase, and RNase H activities. METHODS: HBV Polymerase mutants were transfected into HuH-7 cells and assayed for replication and HBc de-P by the Phos-tag gel analysis. Infection assay was performed by using a HepG2-NTCP-AS2 cell line. RESULTS: Here, we show that a novel phosphatase activity responsible for HBc de-P can be mapped to the C-terminal domain of the polymerase overlapping with the RNase H domain. Surprisingly, while HBc de-P is crucial for viral infectivity, it is essential for neither viral DNA synthesis nor virion secretion. The potential origin, significance, and mechanism of this polymerase-associated phosphatase activity are discussed in the context of an electrostatic homeostasis model. The Phos-tag gel analysis revealed an intriguing pattern of "bipolar distribution" of phosphorylated HBc and a de-P HBc doublet. CONCLUSIONS: It remains unknown if such a polymerase-associated phosphatase activity can be found in other related biosystems. This polymerase-associated phosphatase activity could be a druggable target in clinical therapy for hepatitis B.
Assuntos
Capsídeo , Vírus da Hepatite B , Vírus da Hepatite B/genética , Capsídeo/metabolismo , Montagem de Vírus/genética , DNA Viral , RNA Viral/metabolismo , Proteínas do Capsídeo/metabolismo , Replicação Viral/genética , Ribonuclease H/metabolismo , Monoéster Fosfórico Hidrolases/metabolismoRESUMO
A string of nucleotides confined within a protein capsid contains all the instructions necessary to make a functional virus particle, a virion. Although the structure of the protein capsid is known for many virus species1,2, the three-dimensional organization of viral genomes has mostly eluded experimental probes3,4. Here we report all-atom structural models of an HK97 virion5, including its entire 39,732 base pair genome, obtained through multiresolution simulations. Mimicking the action of a packaging motor6, the genome was gradually loaded into the capsid. The structure of the packaged capsid was then refined through simulations of increasing resolution, which produced a 26 million atom model of the complete virion, including water and ions confined within the capsid. DNA packaging occurs through a loop extrusion mechanism7 that produces globally different configurations of the packaged genome and gives each viral particle individual traits. Multiple microsecond-long all-atom simulations characterized the effect of the packaged genome on capsid structure, internal pressure, electrostatics and diffusion of water, ions and DNA, and revealed the structural imprints of the capsid onto the genome. Our approach can be generalized to obtain complete all-atom structural models of other virus species, thereby potentially revealing new drug targets at the genome-capsid interface.
Assuntos
Bacteriófagos , Capsídeo , DNA Viral , Genoma Viral , Vírion , Montagem de Vírus , Bacteriófagos/química , Bacteriófagos/genética , Bacteriófagos/crescimento & desenvolvimento , Bacteriófagos/metabolismo , Capsídeo/química , Capsídeo/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Difusão , DNA Viral/química , DNA Viral/genética , DNA Viral/metabolismo , Íons/análise , Íons/química , Íons/metabolismo , Eletricidade Estática , Vírion/química , Vírion/genética , Vírion/metabolismo , Montagem de Vírus/genética , Água/análise , Água/química , Água/metabolismoRESUMO
Lentiviral vector (LVV) has been used as one of the common carriers for gene therapy in clinical trials. LVV-mediated clinical trials have being reported in successfully treating hundreds of ß-thalassemia cases. These LVVs bear an inversely placed ß-hemoglobin (HBB) gene expression cassette for preserving introns during the viral RNA packaging. Consequently, these LVVs often produce a small amount of negatively orientated transcript driven by its internal gene promoter and would lower the viral titer by the minus-strand complemented with the viral backbone. To overcome this problem, we designed shRNAs specifically target the minus-strand RNA driven by the LVV internal promoter that resulted in a notable increase in the viral titer. This report demonstrates a simple and positive mean for increasing the effectiveness for gene therapy with the LVV system.
Assuntos
Vetores Genéticos , Lentivirus , RNA Interferente Pequeno , Montagem de Vírus , Lentivirus/genética , Vetores Genéticos/genética , Humanos , RNA Interferente Pequeno/genética , Montagem de Vírus/genética , Células HEK293 , Terapia Genética/métodos , RNA Viral/genética , Carga Viral , Regiões Promotoras Genéticas , Globinas beta/genética , Talassemia beta/terapia , Talassemia beta/genéticaRESUMO
DVGs (Defective Viral Genomes) are prevalent in RNA virus infections. In this investigation, we conducted an analysis of high-throughput sequencing data and observed widespread presence of DVGs in SARS-CoV-2. Comparative analysis between SARS-CoV-2 and diverse DNA viruses revealed heightened susceptibility to damage and increased sequencing sample heterogeneity within the SARS-CoV-2 genome. Whole-genome sequencing depth variability analysis exhibited a higher coefficient of variation for SARS-CoV-2, while DVG analysis indicated a significant proportion of recombination sites, signifying notable genome heterogeneity and suggesting that a large proportion of assembled virus particles contain incomplete RNA sequences. Moreover, our investigation explored the sequencing depth and DVG content differences among various strains. Our findings revealed that as the virus evolves, there is a notable increase in the proportion of intact genomes within virus particles, as evidenced by third-generation sequencing data. Specifically, the proportion of intact genome in the Omicron strain surpassed that of the Delta and Alpha strains. This observation effectively elucidates the heightened infectiousness of the Omicron strain compared to the Delta and Alpha strains. We also postulate that this improvement in completeness stems from enhanced virus assembly capacity, as the Omicron strain can promptly facilitate the binding of RNA and capsid protein, thereby reducing the exposure time of vulnerable virus RNA in the host environment and significantly mitigating its degradation. Finally, employing mathematical modeling, we simulated the impact of DVG effects under varying environmental factors on infection characteristics and population evolution. Our findings provide an explanation for the close association between symptom severity and the extent of virus invasion, as well as the substantial disparity in population infection characteristics caused by the same strain under distinct environmental conditions. This study presents a novel approach for future virus research and vaccine development.